Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
  • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
  • C12Q1/045—Culture media therefor
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
  • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
  • C12Q1/10—Enterobacteria

Definitions

• the present invention relates to a chromogenic culture medium intended for identifying enterococci.
• enterococci In the clinical field, the detection of enterococci is very important because of the appearance of strains which are resistant to antibiotics, in particular to vancomycin, the antibiotic most widely used for treating infections. These nosocomial infections can endanger the lives of the patients affected.
• enterococci A very good study on enterococci can be found at the site www.bacterio.cict.fr/bacdico/ee/enterococcus.html, or at the site www.life.umd.edu/classroom/bsci424/PathogenDescriptions/Enterococcus.htm, or at the site www.enterococcus.ouhsc.edu/lab methods.asp.
• Enterococci generally grow in temperature ranges from 10° to 45° C., the optimum growth being around 35° C., on non-selective medium (blood agar or chocolate agar). However, such a medium also allows the growth of other bacteria and does not make it possible to effectively distinguish enterococci.
• This medium also contains other growth inhibitors such as bile salts. These other inhibitors are effective for inhibiting the growth of Gram-positive bacteria, but not enterococci.
• enterococcal bacteria can also grow in media containing bile salts, the colonies not dissolving after exposure to bile. It is also possible to grow enterococcal bacteria in a medium containing an NaCl concentration of 6.5%, the streptococci not possessing this property.
• Crystal Violet is an inhibitor of enterococcal growth. This hypothesis is reinforced by the fact that among the various MacConkey media, the one used to detect enterococci does not contain Crystal Violet, but contains bile salts. However, this medium also allows the growth of staphylococci.
• the selective effect may be reinforced by incubating the Slanetz and the Bartley medium at 41° C. Most often, complete identification is not obtained and only the identification of catalase is carried out in order to eliminate certain staphylococcal strains which are capable of developing on the media used.
• the prior art media in particular the MacConkey Agar medium sold by Difco Laboratories
• a Crystal Violet concentration equal to 1 mg/l.
• the addition of Crystal Violet at a concentration of 0.1 up to about 1.5 mg/l makes it possible to maintain the growth of enterococci, in particular when the medium does not contain other growth inhibitors for Gram-positive bacteria.
• Crystal Violet makes it possible to avoid using sodium azide which is very often present in enterococci detection media (for its properties of inhibiting the growth of certain bacteria, but which is toxic for humans).
• a medium according to the invention will not therefore contain sodium azide.
• Crystal Violet concentration may be greater than 1 mg/l under certain conditions while allowing the growth of enterococci, could not have been predicted in the light of the knowledge described in the prior art, in particular the fact that it is also possible to eliminate sodium azide.
• the subject of the present invention is a culture medium for detecting and/or distinguishing enterococci, characterized in that it contains, in a culture medium for enterococci, Crystal Violet at a concentration allowing the growth of enterococci and the inhibition of the growth of most Gram-positive bacteria.
• said concentration is greater than 0.1 mg/l, most preferably greater than 0.25 mg/l, or greater than 0.5 mg/l.
• concentration is greater than 0.1 mg/l, most preferably greater than 0.25 mg/l, or greater than 0.5 mg/l.
• said concentration is less than 1.5 mg/l, more preferably less than 1 mg/l.
• the Crystal Violet concentration is about 1 mg/l or greater than this value, it is then advisable to reduce, or even eliminate the other inhibitors of Gram-positive bacteria, in particular the bile salts. It is within the capability of persons skilled in the art to adjust the concentrations of inhibitors according to the concentration of Crystal Violet added to the medium according to the invention.
• the Crystal Violet concentration is less than 0.8 mg/l, more preferably less than 0.7 mg/l. At such concentrations, it is possible to optionally add other inhibitors of Gram-positive bacteria.
• culture medium for enterococci means, that is to say a culture medium containing the nutrients necessary to allow the growth of these bacteria.
• peptone from casein, from soybean, from meat, from yeast extract or from beef, dextrose and the like.
• the medium according to the invention further comprises at least one chromogenic agent, a substrate for an enzyme for fermenting sugars, said enzyme being preferably a glucosidase, in particular ⁇ -glucosidase or a galactosidase, in particular ⁇ -galactosidase.
• said chromogenic agent releases, by hydrolysis, a precipitable chromophore chosen from indoxyl, haloindoxyl (bromoindoxyl, chloroindoxyl, fluoroindoxyl, iodoindoxyl, dichloroindoxyl, chlorobromoindoxyl, trichloroindoxyl), methylindoxyl or hydroxyquinoline derivatives, in particular the following derivatives: 6-chloroindoxyl, 5-bromoindoxyl, 3-bromoindoxyl, 6-fluoroindoxyl, 5-iodoindoxyl, 4,6-dichloroindoxyl, 6-7-dichloroindoxyl, 5-bromo-4-chloroindoxyl, 5-bromo-6-chloroindoxyl, 4,6,7-trichloroindoxyl, N-methylindoxyl or 8-hydroxyquinoline.
• a precipitable chromophore chosen from indoxyl, halo
• said chromogenic substrate for ⁇ -glucosidase is an indoxylglucoside, in particular 5-bromo-4-chloro-3-indoxyl- ⁇ -glucoside and/or said chromogenic agent, a substrate for ⁇ -galactosidase, is an indoxylgalactoside, in particular 5-bromo-6-chloro-3-indoxyl- ⁇ -galactoside.
• growth inhibitors for Gram-negative bacteria such as nalidixic acid or colistin.
• Vancomycin will thus be added at a concentration of about 6 mg/l. It should be noted that it is possible to detect the proportion of resistant bacteria in a sample by plating on a dish without antibiotics and a dish containing them and by comparing the number of colonies identified as enterococci on each dish.
• the invention also relates to the use of a culture medium according to the invention for detecting and/or distinguishing enterococci, and to a method for detecting and/or distinguishing enterococci in a sample, characterized in that it comprises the steps consisting in:
• enterococci is detected by the growth of the colonies on the medium, and this is helped by their coloration after releasing the chromophore from the substrate chromogen for the enzyme.
• a chromophore would be preferably chosen which has a wavelength different from the wavelength of Crystal Violet so as to identify it more easily.
• the medium according to the invention also allows the use and the detection of chromophores having a wavelength close to the wavelength of Crystal Violet.
• the invention also relates to a culture medium for the detection of Gram-positive or Gram-negative bacteria comprising, in addition to growth factors for said Gram-positive or Gram-negative bacteria, a chromogenic agent and Crystal Violet, the Crystal Violet being present at a concentration allowing the growth of said bacteria which it is sought to detect and the differential inhibition of the growth of Gram-positive bacteria, said concentration being preferably between 0.1 and 1.5 mg/l.
• the invention also relates to the use of Crystal Violet as a growth-selective inhibitor for the preparation of a culture medium for the detection of Gram-positive or Gram-negative bacteria, additionally containing a chromogenic agent.
• the invention has therefore demonstrated that it is possible to add a colorant to a chromogenic medium, while retaining the chromogenic properties.
• the chromogen is chosen such that the chromophore released has a wavelength different from that of Crystal Violet or the colorant used in the chromogenic medium.
• a preferred medium for carrying out the invention comprises (for one liter): Agar 15 g Yeast extract and peptones 9 g NaCl 5 g Nalidixic acid 50 mg Colistin 5 mg Crystal Violet 0.5 mg 5-bromo-4-chloro-3-indoxyl- ⁇ -glucoside 50 mg
• the use of the medium according to the invention therefore makes it possible to detect the enterococci, and to distinguish them from staphylococci.
• Examples of media for enterococci which may be used in the context of the present invention, by adding Crystal Violet thereto and by optionally removing sodium azide therefrom, or by adding chromogenic agents, and optionally vancomycin, for detecting resistant strains.
• Bile-esculin medium (composition in grams by liter): Meat extract: 3.0 Meat peptone: 5.0 Beef bile: 40.0 Esculin: 1.0 Iron citrate: 0.5 Agar: 14.5
• This medium may be enriched with 5% horse serum.
• Bile-esculin-azide medium (composition in grams by liter): Tryptone: 17.0 Peptone: 3.0 Yeast extract: 5.0 Esculin: 1.0 NaCl: 5.0 Ammoniacal iron citrate: 0.5 Sodium citrate: 1.0 Sodium azide: 0.25 Beef bile: 10.0 Agar: 13.5
• Oxolinic acid-esculin-azide medium or OAA medium composition in grams per liter: Tryptone: 20.0 Yeast extract: 5.0 Glucose: 1.0 NaCl: 5.0 Ammoniacal iron citrate: 0.5 Sodium citrate: 1.0 Sodium azide: 0.4 Oxolinic acid: 0.005 Agar: 10.0
• Esculin-azide-kanamycin agar composition in grams per liter
• Tryptone 20.0
• Yeast extract 5.0
• Glucose 1.0 NaCl: 5.0
• Ammoniacal iron citrate 0.5
• Sodium citrate 1.0
• Sodium azide 0.15
• Kanamycin sulfate 0.02
• Agar 10.0

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• 2001
  • 2001-06-13 FR FR0107730A patent/FR2826019B1/fr not_active Expired - Fee Related
• 2002
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